Molecular Analysis of ABCR Mutations

Advances in molecular genetics have led to the discovery and identification of genes and genetic mutations that are linked to various visual diseases. Recently, human genetic studies have correlated mutated forms of a retina-specific gene called the ATP binding cassette protein (ABCR) with several inherited visual diseases, including Stargardt’s macular dystrophy, fundus flavimaculatus, age-related macular degeneration, retinitis pigmentosa, and cone-rod dystrophy. The ABCR protein is a transporter protein that plays an important role in retinal rod and cone cells, where it is believed to function in the transport of retinaldehyde. Many of the disease-associated mutations of ABCR have been localized within the ATP-binding cassettes called NBD1 & NBD2. Very little is known regarding the energy transduction process mediated by these two domains. Dr. Biswas is testing the hypothesis that domains NBD1 and NBD2 interact and that this interaction influences ABCR function, particularly ATP hydrolysis. Through the use of cloning and the in-vitro expression of the two halves of the ABCR molecule as individual polypeptides, she is examining the structure and function of each half of the protein. It is hoped that these highly focused molecular/biochemical studies will facilitate better methods of disease diagnosis and treatment, as well as the ability to provide more accurate prognoses of disease development.

Stargardt’s disease and its adult-onset variant, fundus flavimaculatus (FFM), are autosomal recessive disorders affecting approximately 1 in 10,000 persons. These diseases are characterized by a progressive loss of central vision and atrophy of the retinal pigment epithelium, ultimately leading to blindness. Histopathological studies in patients with Stargardt disease and FFM show an accumulation of a lipofuscin-like material within retinal pigment epithelium cells. Clinically arid histopathologically, these symptoms are similar to those observed with age-related macular degeneration.

The retina specific ABC transporter (ABCR) protein plays an important role in retinal rod and cone cells where it is believed function in the transport of retinaldehyde. The ABCR protein is characterized by two nucleotide binding domains and two transmembrane domains, each consisting of six membrane-spanning helices.

Many of the disease-associated mutations of ABCR have been localized within the ATP binding cassettes (NBD1 & NBD2). Very little is known regarding the energy transduction process mediated by these two domains, nor is it known how the ABCR mutations observed in inherited visual disease affect the mechanism of action of this protein. Genetic studies suggest that mutations in the ATP binding cassettes of ABCR lead to defects in ATP hydrolysis, which in turn influence its cellular transport function.

In this proposal, we seek to explore the interaction of NBD1 and NBD2 domains, which appear to play a significant role in the fill1ction of many ABC transporters. The proposed experiments will test the hypothesis that domains NBD1 and NBD2 interact and that this interaction influences ABCR function, in particular ATP hydrolysis. We will further explore the effect of disease-associated mutations on nucleotide-binding domain interaction and nucleotide hydrolysis. In this proposal, we will develop a system for investigating the mechanism of action of human ABCR and structure-function analysis of the biochemical basis of inherited disorders arising from mutations in the ABCR gene. Through cloning and expressing the two halves of the ABCR molecule as individual polypeptides, we will be able to examine the structure and function of each half, something that would not be possible using the full-length native protein. Constructs can then be generated by in vitro mutagenesis that harbors mutations previously shown to be associated with the inherited visual disease. In vitro biochemical and structural studies of these proteins will allow us to understand the consequences of these mutations at a molecular level. The studies that we propose allow us to test the hypothesis that the mutations observed in patients with Stargardt’s and ARMD affect the mediated energy transduction process. Our studies will produce a clearer understanding of the energy transduction process of ATP-dependent transport by the ABCR protein.